(19)
(11)EP 3 300 742 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
04.11.2020 Bulletin 2020/45

(21)Application number: 17200065.5

(22)Date of filing:  08.06.2015
(51)International Patent Classification (IPC): 
A61K 38/40(2006.01)
A61K 31/16(2006.01)
A61K 31/4196(2006.01)
A61P 9/10(2006.01)
A61P 39/04(2006.01)
A61K 31/223(2006.01)
A61K 31/472(2006.01)
A61K 45/06(2006.01)
A61K 31/198(2006.01)
A61K 31/4412(2006.01)
A61P 41/00(2006.01)
A61K 31/4704(2006.01)
A61K 31/426(2006.01)
A61P 25/28(2006.01)

(54)

TRANSFERRIN FOR USE IN THE TREATMENT OF HYPOXIA INDUCIBLE FACTOR (HIF)-RELATED CONDITIONS LIKE ISCHEMIA

TRANSFERRIN ZUR VERWENDUNG BEI DER BEHANDLUNG VON HYPOXIEINDUZIERTEN FAKTOR (HIF)-VERMITTELTEN LEIDEN WIE ISCHÄMIE

TRANSFERRINE DESTINÉE À ÊTRE UTILISÉE DANS LE TRAITEMENT D'AFFECTIONS LIÉES AU FACTEUR INDUCTIBLE PAR L'HYPOXIE (HIF) COMME L'ISCHÉMIE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 11.07.2014 US 201462023446 P

(43)Date of publication of application:
04.04.2018 Bulletin 2018/14

(60)Divisional application:
20197590.1

(62)Application number of the earlier application in accordance with Art. 76 EPC:
15171056.3 / 2977056

(73)Proprietor: Grifols Worldwide Operations Limited
Dublin 22 (IE)

(72)Inventors:
  • ROSS, DAVID A.
    CARY, NC North Carolina 27511 (US)
  • CRUMRINE, RALPH CHRISTIAN
    DURHAM, NC North Carolina 27713 (US)

(74)Representative: Durán-Corretjer, S.L.P. 
Còrsega, 329 (Paseo de Gracia/Diagonal)
08037 Barcelona
08037 Barcelona (ES)


(56)References cited: : 
WO-A1-2008/113134
WO-A1-2014/100233
US-A1- 2006 039 995
US-A1- 2007 148 140
WO-A1-2013/068504
US-A- 6 004 986
US-A1- 2007 092 500
US-B1- 6 251 860
  
  • DE VRIES B ET AL: "Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury", TRANSPLANTATION, WILLIAMS AND WILKINS, GB, vol. 77, no. 5, 15 March 2004 (2004-03-15) , pages 669-675, XP002689444, ISSN: 0041-1337, DOI: 10.1097/01.TP.0000115002.28575.E7
  • MATSUBARA MUNEAKI ET AL: "ABSTRACTS - Myocardial Ischemia and Infarction; Glucagon-like-peptide-1 fused to transferrin: A novel approach to myocardial reperfusion injury", JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, ELSEVIER, NEW YORK, NY, US, vol. 49, no. 9, Suppl. A, 1 March 2007 (2007-03-01), page 236A, XP002471737, ISSN: 0735-1097
  • GOMME P T ET AL: "Transferrin: structure, function and potential therapeutic actions", DRUG DISCOVERY TODAY, ELSEVIER, RAHWAY, NJ, US, vol. 10, no. 4, 15 February 2005 (2005-02-15), pages 267-273, XP027685062, ISSN: 1359-6446 [retrieved on 2005-02-15]
  • ELENA T ZAKHAROVA ET AL: "Human apo-lactoferrin as a physiological mimetic of hypoxia stabilizes hypoxia-inducible factor-1 alpha", BIOMETALS, KLUWER ACADEMIC PUBLISHERS, BO, vol. 25, no. 6, 22 September 2012 (2012-09-22), pages 1247-1259, XP035137893, ISSN: 1572-8773, DOI: 10.1007/S10534-012-9586-Y
  • VON BONSDORFF L ET AL: "Development of a pharmaceutical apotransferrin product for iron binding therapy", BIOLOGICALS, ACADEMIC PRESS LTD., LONDON, GB, vol. 29, no. 1, 1 March 2001 (2001-03-01), pages 27-37, XP002567034, ISSN: 1045-1056, DOI: 10.1006/BIOL.2001.0273 [retrieved on 2002-03-12]
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

FIELD OF THE INVENTION



[0001] The present invention relates to methods of treatment of Hypoxia Inducible Factor (HIF)-related conditions, and in particular to methods of treatment of HIF-related conditions comprising the administration of a composition comprising transferrins.

BACKGROUND OF THE INVENTION



[0002] Protecting cells, and specially neurons, from damage caused by various factors, including stroke, neurodegenerative disease, traumatic injury, etc., is important for longterm recovery of cell or neuronal function. Therapeutic treatment of injured cells or neurons by single agents has advantages, but is often not sufficient to mobilize the complexity of molecules needed to help in restoring complete function.

[0003] Physiological response to protect neurons or other cells from hypoxic or ischemic events, or from oxidation, is often considered to be mediated by expression of genes that are up-regulated through the Hypoxia Inducible Factor (HIF) signaling pathway, a key regulatory pathway that is responsive to cellular insults. In the brain, upregulation of neuroprotective molecules is believed to be a critical factor in protecting cells from irreparable damage. However, few available drugs are sufficiently able to prevent, restore or reduce damage to neurons and other tissues. Additionally they are often toxic, have short half-lives, or both. For example, the international patent application WO2006/20727 proposes the use of deferoxamine as neuroprotector agent against the harmful effects of reperfusion; however, the administration of deferoxamine poses problems due to its reduced half-life in plasma.

[0004] Transferrins are iron-binding blood plasma glycoproteins that control the level of free iron in biological fluids. Transferrins function as the main transporter of iron in the circulation where it exists in an iron-free apo-transferrin (ApoTf) form, as monoferric transferrin, or as diferric holo-transferrin (HoloTf). Typically, iron is bound to 30% of all transferrin binding sites in circulation. The neuroprotection function of ApoTf but not HoloTf has been disclosed by Chen-Roetling et al. (Chen-Roetling J., Chen L., and Regan R.F. Neuropharmacology, 2011; 60(2-3): 423-431.), suggesting that ApoTf may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage.

[0005] The present inventors have found that it may be possible to boost the neuroprotective properties of transferrin administration in patients by combining it with other iron chelating agents or with another iron-binding plasma protein, such as Apolactoferrin, which has been shown to increase HIF-1α protein levels in some tissues and have effects on plasma EPO levels (Zakharova E.T. et al. Biometals (2012) 25:1247-1259). Molecules with iron chelating abilities have been suggested to be HIF pathway activators by blocking the activity of prolyl hydroxylases.

[0006] Therefore, the present invention refers to a composition comprising a mixture of apo-transferrin (Apo-Tf) and holo-transferrin (Holo-Tf) for use in treatment of ischemia or oxygen deprivation in a patient prior to surgery, ischemia due to cardiac arrest, ischemia due to thrombotic clots, or ischemia due to traumatic injury wherein said composition is a mixture of apo-transferrin (Apo-Tf) and holo-transferrin (Holo-Tf), in a ratio of 98% Apo-Tf:2% Holo-Tf to 30% Apo-Tf:70% Holo-Tf.

BRIEF DESCRIPTION OF THE DRAWINGS



[0007] The present invention is further described with reference to the following drawings, in which:

Figure 1 shows that compositions of majority ApoTf and majority HoloTf induce HIF-1α protein under normoxic and hypoxic conditions 6 hrs post treatment.

Figure 2 shows that compositions of majority ApoTf and majority HoloTf induce HIF-1α protein under normoxic conditions 24 hrs post treatment.

Figure 3 shows that mixtures of ApoTf and HoloTf induce HIF-1α protein 6 hrs post treatment.

Figure 4A shows mRNA expression levels of Glut1 under normoxic and hypoxic conditions in the presence of HSA, Apo-transferrin or Holo-transferrin.

Figure 4B shows mRNA expression levels of VEGF under normoxic and hypoxic conditions in the presence of HSA, Apo-transferrin or Holo-transferrin.

Figure 5A shows in vitro or in vivo toxicity of compositions comprising either majority HoloTf or majority ApoTf.

Figure 5B shows Modified Bederson and General Behavioral scores for rats intravenously treated with drug comprising majority of ApoTf or HoloTf.

Figure 6A shows Scatter plot of the infarct volume of ApoTf (385 mg/kg, IV) or saline treatment in transient Middle Cerebral Artery occlusion (MCAo) rat model.

Figure 6B shows triphenyltetrazolium chloride (TTC) stained Coronal sections from a representative control and ApoTf treated rat.

Figure 7 shows protection of neuronal cells from the toxic effects of Abeta(1-42) by mixtures comprising mostly ApoTf and HoloTf.

Figure 8A shows treatment of SH-SY5Y neuronal cells with 4 mg/ml of majority ApoTf and with a combination of majority ApoTf and DFO or IOX2.

Figure 8B shows treatment of SH-SY5Y neuronal cells with 4 mg/ml of the indicated protein and with a combination of the indicated protein and 10uM M30 plus.

Figure 8C shows treatment of SH-SY5Y neuronal cells with 4 mg/ml of the indicated protein and with a combination of the indicated protein and 200uM DFO.

Figure 9A shows mRNA expression levels of Glut1 in response to majority Apotransferrin and DFO or IOX2 combinations.

Figure 9B shows mRNA expression levels of VEGF in response to majority Apotransferrin and DFO or IOX2 combinations.

Figure 10A shows HIF-1α levels after treatment of Primary human renal proximal tubule epithelial (RPTEC) cells with 4 mg/mL majority Apo-transferrin, majority Holo-transferrin or various mixtures of each for 6hrs under normal oxygen levels.

Figure 10B shows HIF-1α levels after treatment of primary cortical epithelial (HRCE) cells with 4 mg/mL majority Apo-transferrin, majority Holo-transferrin or various mixtures of each for 6hrs under normal oxygen levels.

Figure 11A shows viability of primary human renal proximal tubule epithelial (RPTEC) or cortical epithelial (HRCE) cells when treated with majority ApoTf or DFO for 48 hours.

Figure 11B shows viability of RPTEC or HRCE cells when treated for 72hrs with 4mg/mL of majority ApoTf, majority HoloTf, mixtures of transferrin.

Figure 12 shows caspase 3/7 activation within human primary kidney cells in the presence of majority ApoTf or DFO.

Figure 13A shows HIF-1α levels after treatment of lung cell line NCI-H1650 with 4 mg/mL majority Apo-transferrin, majority Holo-transferrin or pd-Transferrin for 6hrs under normal oxygen levels.

Figure 13B shows HIF-1α levels after treatment of primary hepatocyte cells with 4 mg/mL majority Apo-transferrin, majority Holo-transferrin or pd-Transferrin for 6hrs under normal oxygen levels.

Figure 14A shows viability of human lung cell line, NCI-H1650, when treated for 72 hours with 4mg/mL of majority ApoTf, majority HoloTf, or pd- transferrin.

Figure 14B shows viability of primary human hepatocytes when treated for 72 hours with 4mg/mL of majority ApoTf, majority HoloTf, or pd- transferrin.


DETAILED DESCRIPTION OF THE INVENTION



[0008] The present invention refers to a composition comprising a mixture of apo-transferrin (Apo-Tf) and holo-transferrin (Holo-Tf) for use in treatment of ischemia or oxygen deprivation in a patient prior to surgery, ischemia due to cardiac arrest, ischemia due to thrombotic clots, or ischemia due to traumatic injury wherein said composition is a mixture of apo-transferrin (Apo-Tf) and holo-transferrin (Holo-Tf), in a ratio of 98% Apo-Tf:2% Holo-Tf to 30% Apo-Tf:70% Holo-Tf.

[0009] Preferably, said composition further comprising either an iron chelator or PHD2 enzyme inhibitor.

[0010] Preferably, in said composition said Apo-Tf and Holo-Tf are recombinant.

[0011] Preferably, said Apo-Tf and Holo-Tf are modified by pegylation, glycosylation, polysialylation, or other physical modifications to extend plasma half-life of the protein, including covalent fusion to domains that extend half-life in blood, such as the Fc domain of immunoglobulin, albumin, XTEN.

[0012] Also preferably, said Apo-Tf and Holo-Tf are protein conjugates between full length Apo-Tf and Holo-Tf or fragments of Apo-Tf and Holo-Tf with any other protein, protein fragment, or peptide.

[0013] Preferably, said Apo-Tf and Holo-Tf are derivatives of transferrin comprising more than 50% similarity to SEQ ID NO:1.

[0014] More preferably, said iron chelator is M30, deferoxamine (DFO), Deferasirox, deferiprone, deferitrin, L1NAII, CP363, CP502, or Ethylenediaminetetraacetic acid (EDTA).

[0015] Even more preferably, said PHD2 enzyme inhibitor is IOX2, IOX3, dimethyloxallyl glycine or other 2-oxoglutarate binding site molecules.

[0016] When transferrin is recombinant, it can be obtained according to any technique known in the art of protein expression, production and purification. For example, nucleic acid sequence of transferrin can be inserted in any vector suitable for expression in the elected host cell, e.g. bacteria (Escherichia coli, Bacilus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces and Staphylococcus), yeast (Saccharomyces, Pichia or Kuyveromyces genus), insect cells (Bombyx mori, Mamestra brassicae, Spodoptera frugiperda, Trichoplusia ni or Drosophila melanogaster) or mammalian cells (HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), human adenovirus transformed 293 cells, mouse L-929 cells, HaK hamster cell lines, murine 3T3 cells derived from Swiss, Balb-c or NIH mice, CV-1 cell line, cell strains derived from in vitro culture of primary tissue or primary explants).

[0017] It is contemplated that plasma derived transferrin is isolated from a suitable fraction of plasma. In a preferred embodiment transferrin is isolated from fraction IV, and most preferably fraction IV-I or fraction IV-IV, of the Cohn fractionation process. In another preferred embodiment, transferrin derives from a waste fraction of a method of purifying alpha1-proteinase inhibitor (A1PI). Said purifying method can be as follows:
  1. (a) removing a portion of contaminating proteins from the aqueous solution by precipitation in order to obtain a purified solution containing A1 PI;
  2. (b) passing the purified solution through an anion exchange resin so that A1 PI binds to the anion exchange resin;
  3. (c) eluting A1 PI from the anion exchange resin to obtain an eluted solution containing A1 PI;
  4. (d) passing the eluted solution through a cation exchange resin;
  5. (e) collecting a flow-through from the cation exchange resin that contains A1PI; and
  6. (f) contacting the eluted solution of step (c) or the flow-through of step (e) with a hydrophobic adsorbent of at least one HIC medium.


[0018] The aqueous solution used in the method of purifying (A1 PI) mentioned above can be blood, plasma or a plasma derived fraction.

[0019] A person skilled in the art would envisage that the residues or zones to be modified can be determined by several techniques know in the art as, for example, site directed mutagenesis, alanine screening, crystallography or analysis of deletions and/or insertions.

[0020] It is contemplated that transferrin of the present invention is in the form of a protein conjugate or a fusion protein in order to, for example, extend its half-life in blood, wherein transferrin is conjugated or fused to any other protein, protein fragment, protein domain or peptide. In a preferred embodiment, transferrin is conjugated or fused to a full-length, fragment, domain or peptide of serum albumins (as for example, bovine, from rabbits or from humans), keyhole limpet hemocyanin, immunoglobulin molecules (including Fc domain of immunoglobulins), thyroglobulin, ovalbumin, or XTEN.

[0021] While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

EXAMPLES



[0022] Experiments performed in the following examples were treated with transferrin comprising either the Apo- or Holo- forms. A broad variety of transferrin mixtures were tested; the relative percentages of ApoTf and HoloTf comprising majority ApoTf, majority HoloTf, pdTf and specifically defined transferrin mixtures are highlighted in Table 1 below.
Table 1:
 MajorityMajority    
 ApoHoloMix 1Mix 2Mix 3pdTf
% ApoTf 98 30 95 64 33 68
% HoloTf 2 70 5 36 67 32

Example 1. Compositions comprising majority ApoTf and majority HoloTf induce HIF-1α protein under normoxic and hypoxic conditions after 6 hours of treatment.



[0023] Human neuroblastoma SH-SY5Y cell line cells, cultured in serum free media were treated with plasma derived ApoTf and HoloTf (in both cases at a concentration of 1mg/mL and 4mg/mL) for 6 hours under normoxia (21% oxygen) and hypoxia conditions (1% oxygen). As controls, untreated cells or cells treated with human serum albumin (HSA) at a concentration of 1mg/mL or 4mg/mL, are used. After 6hrs intracellular proteins were harvested and tested for HIF-1α protein levels by ELISA.

[0024] As shown in Figure 1, a significant increase in HIF-1α cellular protein levels occurred under both normoxic and hypoxic conditions and for the two concentrations tested for ApoTf. Regarding HoloTf a significant increase in the cellular protein levels of HIF-1α was observed for both normoxic and hypoxic conditions when cells were treated with the 1mg/mL concentration and for normoxic condition when cells were treated with the 4mg/mL concentration. When cells were treated with the 4mg/mL concentration of HoloTf a tendency towards an increase in the cellular protein levels of HIF-1α was seen.

Example 2. Compositions comprising majority ApoTf and majority HoloTf induce HIF-1α protein under normoxic conditions after 24 hours of treatment.



[0025] Experiment performed in example 1 was repeated but performing treatments for 24 hours and only under normoxic conditions. After 24 hrs intracellular proteins were harvested and tested for HIF-1α protein levels by ELISA. Figure 2 shows the results obtained for this experiment. As can be seen in said figure, ApoTf increased cellular protein levels of HIF-1α in both concentrations tested. For HoloTf a significant increase of cellular protein levels of HIF-1α was observed when treatment was performed using a concentration of 4mg/mL and a tendency towards an increase of said protein was seen when using the 1mg/mL concentration.

Example 3. Mixtures of ApoTf and HoloTf induce HIF-1α protein after 6 hours of treatment.



[0026] After 6 hrs, intracellular proteins were harvested and tested for HIF-1α protein levels by ELISA. As shown in Figure 3, an increase of HIF-1α cellular protein levels after 6 hours treatment with plasma derived majority ApoTf, majority HoloTf or mixtures thereof under normoxic conditions was observed. As also can be seen in said figure, all mixtures of ApoTf and HoloTf upregulate HIF-1α protein in SH-SY5Y neuronal cells.

Example 4. mRNA expression levels of Glut1 and VEGF under normoxic and hypoxic conditions in the presence of HSA, Apo-transferrin or Holo-transferrin.



[0027] The stabilization of, and increase in, HIF-1α protein typically leads to an upregulation of HIF-related genes (increase in the transcription of genes targeted by HIF), i.e. genes that have HIF binding sites in their transcriptional regulatory elements. Two well characterized genes that are activated by HIF-1α protein are Glut1 receptor and VEGF. Therefore, in order to analyze mRNA expression changes in each of these HIF target genes SH-SY5Y cell line cells were cultured and treated with majority ApoTf or majority HoloTf at a concentration of 1mg/mL and 4mg/mL under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for 6 hours. As a negative controls, cells were treated with HSA (1mg/mL or 4mg/mL) or were left untreated. After 6hrs, intracellular mRNA was harvested and tested for Glut1 and VEGF expression levels by qPCR. Expression results were calculated relative to the expression of the corresponding transcript seen in untreated cells. Figures 4A and 4B show the expression results obtained for Glut1 receptor and VEGF, respectively. Values in the figures are shown as Relative Gene Expression, with the target gene (Glut1 or VEGF) normalized for housekeeper (beta-actin) expression. As can be directly derived from said figures, under hypoxic conditions, expression of both Glut1 (Fig. 4A) and VEGF (Fig. 4B) were significantly increased when treated with Apo-transferrin relative to HSA controls. Interestingly, under normoxic conditions, Holo-transferrin, but not Apo-transferrin, increased expression of only Glut1.

Example 5. Mixtures of ApoTf and HoloTf do not show toxicity in vitro or in vivo.



[0028] Since it has been reported that HoloTf is toxic to cells in vivo and in vitro, toxicity of various compositions containing majority ApoTf, majority HoloTf or mixtures of ApoTf + HoloTf was tested in SH-SY5Y cells. SH-SY5Y cells were treated with the indicated concentrations of 4mg/mL Tf (as indicated in Figure 5A), M30 or DFO for 72 hours. After 72 hours, cells were subjected to a Cell Titer Glow viability assay. Control cells, untreated cells, were set to a value of 100% viable. The average viability and standard deviations are shown for each treatment condition. No toxic effects were seen with any composition containing majority ApoTf and, surprisingly, no toxicity or detrimental effects of majority HoloTf were observed.

[0029] Interestingly, neither compositions of majority ApoTf nor majority HoloTf showed significant differences in these behavioral criteria, suggesting that there were no detrimental effects of HoloTf in vivo. Figure 5B shows modified Bederson and General Behavioral scores for rats intravenously treated with drug comprising majority of ApoTf or HoloTf. In vivo neurological function was assessed by a modified Bederson score (Bederson et al., 1986b; Crumrine et al., 2011) using the following definitions:

Score 0: No apparent neurological deficits;

Score 1: Body torsion present;

Score 2: Body torsion with right side weakness;

Score 3: Body torsion, right side weakness with circling behavior; and

Score 4: Seizure Activity.



[0030] General behavioral scores of rats were developed by the CALS personnel for the purpose of monitoring recovery of animals following surgical procedures (standard CALS post-operative care). A numerical value was assigned to the predetermined behavioral observations.

Score 0: Behavior consistent with a normal naive rat (i.e. no ipsilateral deficit);

Score 1: Bright/active/responsive; the rat spontaneously moves and explores his cage, responds to external stimuli, explores the top of the cage;

Score 2: Quiet/alert/responsive; reserved behavior but will respond to external stimulus, tends not to rear or explore the top of the cage;

Score 3: Depressed behavior: tends not to move unless prodded, quickly returns to a somnolent state, little to no interest in external stimuli;

Score 4: Unresponsive: remains in a prostrate position even when prodded; and

Score 5: Seizure activity requiring euthanasia.


Example 6. In vivo cellular protection by transferrin.



[0031] The MCAo (Middle Cerebral Artery occlusion) rat model of brain stroke was used to assess cellular protection by transferrin. Stroke was surgically induced to 16 rats by using the MCAo technique. 8 rats were treated by injection of saline solution in the brain and the other 8 by injection of ApoTf in the brain. Figures 6A and 6B shows that a significant decrease in the volume of the infracted area was observed in the rats treated with a mixture comprising a majority of ApoTf when compared with control rats (treated with saline solution). Figure 6A shows a scatter plot of the infarct volume of ApoTf (385 mg/kg, IV) or saline treatment in transient MCAo; and Figure 6B shows TTC stained Coronal sections from a representative control and ApoTf treated rat.

Example 7. ApoTf and HoloTf protect SH-SY5Y from Abeta 1-42 toxicity.



[0032] Upregulation of the HIF pathway is known to play a protective role in a number of neurodegenerative diseases, including pathologies that result in destruction of nerve cells and neurons. Since treatment of SH-SY5Y upregulates HIF, treatment of cells with Apo- or Holo-transferrin should provide a protective effect on cells subjected to substances known to induce neurodegeneration. Figure 7 highlights data assessing whether majorities of Apo- and Holo-transferrin could protect SH-SY5Y cells from the toxic effects of the known neurodegenerative toxin oligomerized Abeta 1-42 peptide (Figure 7). SH-SY5Y neuronal cells cultured in growth media were treated with 4 mg/mL Apo-transferrin or Holo-transferrin for 24hrs under normal oxygen levels. After 24hrs, cells were treated with oligomerized Abeta1-42 peptide for an additional 72 hours. Following treatment with oligomerized Abeta1-42, cells were subjected to a ApoGlo caspase 3/7 activation assay. Control cells, untreated cells, were set to a normalized value of 1. The average caspase induction, relative to control cells, and standard deviations are shown for each treatment condition. Interestingly, these data show that both majority ApoTf and HoloTf protect SH-SY5Y cells from Abeta induced toxicity. These data also further confirm lack of inherent toxicity with either ApoTf or HoloTf.

Example 8. Synergystic effect with small molecule HIF activators and ApoTf/HoloTf mixtures.



[0033] Transferrin may act synergistically with other HIF activating small molecules, such as other iron chelators or enzyme inhibitors. This could allow lower levels of these small molecules to be administered, eliciting fewer side effects but retaining high therapeutic levels. To determine whether Apotransferrin increases the potency of the iron chelator, DFO, and the phd2 inhibitor IOX2; SH-SY5Y neuronal cells cultured in serum free media were treated with 4 mg/mL of the indicated proteins in the presence or absence of small molecule drug under normal oxygen levels. The results of the experiment are shown in Figures 8A, 8B, and 8C. The data shown in Figure 8A relates to treatment of cells with a combination of DFO or IOX2, at the indicated concentrations, plus 4mg/mL protein. CoCl2 was used as an experimental positive control. The data shown in Figure 8B relates to treatment of cells with a combination of 10uM M30 plus/minus 4mg/mL protein. The data shown in Figure 8C relates to treatment of cells with a combination of 200uM DFO plus/minus 4mg/mL protein. After 6hrs intracellular proteins were harvested and tested for HIF-1α protein levels by ELISA. Data are shown in pg/mL with standard deviation.

Example 9. mRNA expression levels of Glut1 and VEGF in response to majority Apotransferrin and DFO or IOX2 combinations.



[0034] In addition, mRNA expression levels of Glut1 and VEGF in response to majority Apotransferrin and DFO or IOX2 combinations were determined. SH-SY5Y neuronal cells cultured in serum free media were treated with 4 mg/mL human serum albumin or majority Apotransferrin under normal oxygen levels. Where indicated, either 200uM DFO or 1uM IOX2 were co-treated with the HSA and majority Apotransferrin. After 6hr treatments, intracellular mRNA was harvested and tested for Glut1 and VEGF expression levels by qPCR. Values are shown as Relative Gene Expression, with the target gene (Glut1 or VEGF) normalized for housekeeper (beta-actin) expression. Standard deviations are shown. Figures 9A and 9B show that Glut1 and VEGF mRNA levels increase synergistically and additively with the addition of both Apotransferrin and small molecule activators of the HIF pathway.

Example 10. Compositions of majority ApoTf and majority HoloTf induce HIF-1α protein in human primary kidney cells.



[0035] It is well-known in the art that many small molecules used for the treatment of conditions related or provoked by hypoxia are toxic and have numerous side effects, e.g. DFO. One of the most apparent side effects of said small molecules is kidney toxicity. Therefore, in order to assess whether transferrin and/or mixtures increase HIF-1α levels in primary kidney cells; human primary kidney cells, both primary human renal proximal tubule epithelial (RPTEC) or cortical epithelial cells (HRCE) were obtained. Primary human renal proximal tubule epithelial (RPTEC) or primary cortical epithelial (HRCE) cells cultured in serum free media were treated with 4 mg/mL majority Apotransferrin, majority Holo-transferrin or various mixtures of each for 6hrs under normal oxygen levels. After 6hrs intracellular proteins were harvested and tested for HIF-1α protein levels by ELISA. Figures 10A and 10B reveal that HIF-1α levels are induced with transferrin composed of mixtures of Apo-transferrin and Holo-transferrin in RPTEC and HRCE, respectively.

Example 11. Viability of human primary kidney cells in the presence of Transferrins or DFO, including Caspase 3/7 activation within human primary kidney cells in the presence of majority ApoTf or DFO.



[0036] Considering the anticipated safety profile of a human plasma protein, toxicity of DFO and transferrins (majority Apo, majority Holo and mixtures) was assessed in primary human kidney cells. The renal proximal tubule epithelial (RPTEC) or cortical epithelial (HRCE) cells were treated with the indicated concentrations of majority ApoTf or DFO for 48 hours (Figure 11A); and RPTEC or HRCE cells were treated for 72hrs with 4mg/mL of majority ApoTf, majority HoloTf, mixtures of transferrin (Figure 11B). After 48 or 72 hours, cells were subjected to a Cell Titer Glow viability assay. Control cells, untreated cells, were set to a value of 100% viable. The average viability and standard deviations are shown for each treatment condition. Figures 11A and 11B shows that while DFO had significant toxicity, none of the transferrin molecules showed any detrimental effects on these primary kidney cells.

[0037] In order to assess caspase 3/7 activation within human primary kidney cells in the presence of ApoTf or DFO; RPTE or HRC cells were treated with the indicated concentrations of ApoTf or DFO for 48 hours. After 48 hours, cells were subjected to a ApoGlo caspase 3/7 activation assay. Control cells, untreated cells, were set to a normalized value of 1. The average caspase activity, relative to control cells, and standard deviations are shown in Figure 12 for each treatment condition.

Example 12. No upregulation of HIF was observed in primary human hepatocytes or NCI-H1650, a lung cell line.



[0038] As detailed above, both plasma derived Apo-transferrin and Holo-transferrin increase the cellular levels of HIF-1α, in the human neuronal cell line SH-SY5Y. In addition to neuronal cells, liver and lung organ transplants may also benefit from induction of HIF signaling. Hence, in order to assess the same; effect of transferrins on HIF-1α levels in primary hepatocytes and a lung cell line (NCI-H1650) was determined.

[0039] The lung cell line NCI-H1650 or primary hepatocyte cells cultured in serum free media were treated with 4 mg/mL majority Apo-transferrin, majority Holotransferrin or pd-Transferrin for 6hrs under normal oxygen levels. After 6hrs intracellular proteins were harvested and tested for HIF-1α protein levels by ELISA. The data, as highlighted in Figures 13A and 13B, shows that HIF-1α levels are not induced with transferrin or mixtures of Apo-transferrin and Holo-transferrin in NCIH1650 or primary hepatocytes.

Example 13. Viability of NCI-H1650 and human primary hepatocytes in the presence of Transferrins.



[0040] Given the anticipated safety profile of a human plasma protein, toxicity of transferrins (majority Apo, majority Holo and pd-transferrin) in NCI-H1650 and primary human hepatocyte cells was assessed. The human lung cell line, NCI-H1650, and primary human hepatocytes were treated for 72 hours with 4mg/mL of majority ApoTf, majority HoloTf, or pd- transferrin. After 72 hours, cells were subjected to a Cell Titer Glow viability assay. Control cells, untreated cells, were set to a value of 100% viable. The average viability and standard deviations are shown in Figures 14A and 14B for each treatment condition. The data shows that no toxicity was observed with compositions containing either majority HoloTf or majority ApoTf in lung cells, NCI-H1650, or primary hepatocytes.

CONCLUSIONS:



[0041] The experiments performed in the human neuronal cell line SH-SY5Y showed that both plasma derived Apo-transferrin and Holo-transferrin increased the cellular levels of HIF-1α. The increase in HIF-1α levels occurred under both normoxic and hypoxic conditions. Administration of Apo-transferrin to cells under normal oxygen conditions raised the levels of HIF-1α to a similar level of that seen when cells were exposed to a hypoxic environment. Exposure of SH-SY5Y cells to Apo-transferrin in normoxic conditions for longer periods increased the level of HIF-1α to a greater extent than shorter time. The human serum albumin negative controls had no effect on HIF-1α levels.

[0042] Various mixtures of ApoTf and HoloTf all upregulated HIF-1α protein in SH-SY5Y neuronal cells and primary kidney cells.

[0043] No upregulation of HIF-1α was observed in primary human hepatocytes, or NCI-H1650, a lung cell line.

[0044] Various mixtures of ApoTf and HoloTf all upregulated HIF-1α target genes in SH-SY5Y neuronal cells.

[0045] No toxicity was observed with compositions containing either majority HoloTf or majority ApoTf in any cell type (neuronal, lung, kidney or hepatocyte) or in vivo.

[0046] In vivo treatment of rats in a neurological stress model of ischemia-reperfusion showed that transferrin (composed of mostly ApoTf) protects rat cells from infarct.

[0047] Mixtures comprising mostly of ApoTf or HoloTf protected neuronal cells from the toxic effects of Abeta (1-42) oligomer.

[0048] Only mixtures composed of majority ApoTf had synergistic effects with M30 or DFO, and these synergistic activities only occurred in SH-SY5Y neuronal cells.
Human Transferrin \226 698 amino acids


Sequence Reference:



[0049] 

GENBANK ACCESSION AAB22049

AUTHORS Hershberger,C.L., Larson,J.L., Arnold,B., Rosteck,P.R. Jr.,
Williams,P., DeHoff,B., Dunn,P., O'Neal,K.L., Riemen,M.W., Tice,P.A. et al.

TITLE A cloned gene for human transferrin

JOURNAL Ann. N. Y. Acad. Sci. 646, 140-154 (1991)




Claims

1. Composition comprising a mixture of apo-transferrin (Apo-Tf) and holo-transferrin (Holo-Tf) for use in treatment of ischemia or oxygen deprivation in a patient prior to surgery, ischemia due to cardiac arrest, ischemia due to thrombotic clots, or ischemia due to traumatic injury wherein said composition is a mixture of apo-transferrin (Apo-Tf) and holo-transferrin (Holo-Tf), in a ratio of 98% Apo-Tf:2% Holo-Tf to 30% Apo-Tf:70% Holo-Tf.
 
2. Composition for use according to claim 1, further comprising either an iron chelator or PHD2 enzyme inhibitor.
 
3. Composition for use according to claim 1, wherein said Apo-Tf and Holo-Tf are recombinant.
 
4. Composition for use according to claim 1, wherein said Apo-Tf and Holo-Tf are modified by pegylation, glycosylation, polysialylation, or other physical modifications to extend plasma half-life of the protein, including covalent fusion to domains that extend half-life in blood, such as the Fc domain of immunoglobulin, albumin, XTEN.
 
5. Composition for use according to claim 1, wherein said Apo-Tf and Holo-Tf are protein conjugates between full length Apo-Tf and Holo-Tf or fragments of Apo-Tf and Holo-Tf with any other protein, protein fragment, or peptide.
 
6. Composition for use according to claim 1, wherein said Apo-Tf and Holo-Tf are derivatives of transferrin comprising more than 50% similarity to SEQ ID NO:1.
 
7. Composition for use according to claim 2, wherein said iron chelator is M30, deferoxamine (DFO), Deferasirox, deferiprone, deferitrin, L1NAII, CP363, CP502, or Ethylenediaminetetraacetic acid (EDTA).
 
8. Composition for use according to claim 2, wherein said PHD2 enzyme inhibitor is IOX2, IOX3, dimethyloxallyl glycine or other 2-oxoglutarate binding site molecules.
 


Ansprüche

1. Zusammensetzung, umfassend eine Mischung aus Apo-Transferrin (Apo-Tf) und Holo-Transferrin (Holo-Tf) zur Verwendung bei der Behandlung von Ischämie oder Sauerstoffmangel in einem Patienten vor einer Operation, Ischämie aufgrund von Herzstillstand, Ischämie aufgrund von thrombotischen Gerinnseln oder Ischämie aufgrund von traumatischen Verletzungen, wobei die Zusammensetzung eine Mischung aus Apo-Transferrin (Apo-Tf) und Holo-Transferrin (Holo-Tf) in einem Verhältnis von 98 % Apo-Tf:2 % Holo-Tf bis 30 % Apo-Tf:70 % Holo-Tf ist.
 
2. Zusammensetzung zur Verwendung nach Anspruch 1, ferner umfassend entweder einen Eisen-Chelatbildner oder einen PHD2-Enzym-inhibitor enthält.
 
3. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das Apo-Tf und Holo-Tf rekombinant sind.
 
4. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das Apo-Tf und Holo-Tf durch Pegylierung, Glykosylierung, Polysialylierung oder andere physikalische Modifikationen modifiziert sind, um die Plasmahalbwertszeit des Proteins zu verlängern, einschließlich der kovalenten Fusion an Domänen, die die Halbwertszeit im Blut verlängern, wie die Fc-Domäne eines Immunglobulins, Albumin, XTEN.
 
5. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das Apo-Tf und Holo-Tf Proteinkonjugate zwischen Volllängen-Apo-Tf und Volllängen-Holo-Tf oder Fragmenten von Apo-Tf und Holo-Tf mit irgendeinem anderen Protein, Proteinfragment oder Peptid sind.
 
6. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das Apo-Tf und Holo-Tf Derivate von Transferrin sind, die mehr als 50 % Ähnlichkeit mit SEQ ID NO:1 aufweisen.
 
7. Zusammensetzung zur Verwendung nach Anspruch 2, wobei der Eisenchelatbildner M30, Deferoxamin (DFO), Deferasirox, Deferipron, Deferitrin, L1NAII, CP363, CP502 oder Ethylendiamintetraessigsäure (EDTA) ist.
 
8. Zusammensetzung zur Verwendung nach Anspruch 2, wobei der PHD2-Enzyminhibitor IOX2, IOX3, Dimethyloxallylglycin oder andere Moleküle mit 2-Oxoglutarat-Bindungsstelle sind.
 


Revendications

1. Composition comprenant un mélange de apo-transferrine (Apo-Tf) et de holo-transferrine (Holo-Tf) pour utilisation dans le traitement de l'ischémie ou du manque d'oxygène chez un patient avant la chirurgie, de l'ischémie due à un arrêt cardiaque, de l'ischémie due à des caillots thrombotiques, ou de l'ischémie due à une blessure traumatique, dans laquelle ladite composition est un mélange de apo-transferrine (Apo-Tf) et de holo-transferrine (Holo-Tf), selon un rapport allant de 98 % de Apo-Tf pour 2 % de Holo-Tf à 30 % de Apo-Tf pour 70 % de Holo-Tf.
 
2. Composition pour utilisation selon la revendication 1, comprenant en outre soit un chélateur du fer soit un inhibiteur de l'enzyme PHD2.
 
3. Composition pour utilisation selon la revendication 1, dans laquelle lesdites Apo-Tf et Holo-Tf sont recombinantes.
 
4. Composition pour utilisation selon la revendication 1, dans laquelle lesdites Apo-Tf et Holo-Tf sont modifiées par pégylation, glycosylation, polysialylation, ou autres modifications physiques pour étendre la demi-vie de plasma de la protéine, comprenant une fusion covalente à des domaines qui étendent la demi-vie dans le sang, tels que le domaine Fc de l'immunoglobuline, l'albumine, XTEN.
 
5. Composition pour utilisation selon la revendication 1, dans laquelle lesdites Apo-Tf et Holo-Tf sont des conjugués de protéines entre des Apo-Tf et Holo-Tf en pleine longueur ou des fragments de Apo-Tf et Holo-Tf avec une autre protéine, un fragment de protéine, ou un peptide.
 
6. Composition pour utilisation selon la revendication 1, dans laquelle lesdites Apo-Tf et Holo-Tf sont des dérivés de la transferrine comprenant plus de 50 % de similarité avec SEQ ID NO :1.
 
7. Composition pour utilisation selon la revendication 2, dans laquelle ledit chélateur du fer est M30, la déféroxamine (DFO), le déférasirox, le défériprone, la déféritrine, L1NAII, CP363, CP502, ou l'acide éthylènediaminetétraacétique (EDTA).
 
8. Composition pour utilisation selon la revendication 2, dans laquelle ledit inhibiteur de l'enzyme PHD2 est IOX2, IOX3, la diméthyloxallyl glycine ou d'autres molécules à site de liaison au 2-oxoglutarate.
 




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Cited references

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